Insulin is stored in dense core vesicles (DCVs) in pancreatic beta cells. Release of insulin from a vesicle requires transient fusion of the DCV membrane with the plasma membrane. This is regulated, in part, by phospholipids and transmembrane proteins in the DCV. Phogrin (IA-2beta) is a transmembrane protein tyrosine phosphatase-like protein that is localized to secretory granules. This has suggested that it plays a role in regulating insulin secretion, but a potential catalyitc site of Phogrin contains Asp in place of an expected tyrosine phosphatase consensus Ala, and no enzymatic activity for Phogrin has ever been reported. I have obtained evidence that Phogrin is able to dephosphorylate phosphatidylinositol phospholipids (PIPs), including PI(3)P and PI(4,5)P2. Protein domains that bind PI(3)P and PI(4,5)P2 serve to localize specific proteins to the correct vesicle compartment and to alter the activity of the bound proteins and thus, function to regulate the formation, release, and recycling of secretory vesicles. I will test the hypothesis that Phogrin is a PIPase and that it plays a role in regulating insulin release by regulating PIP levels in secretory vesicles and recycling endosomes. I will test this hypothesis in 4 specific aims: Aim 1. Characterize the PIPase activity of full length Phogrin in vitro. Aim 2. Test pharmacologic inhibitors of the PIPase activity of Phogrin. Aim 3. Test the hypothesis that phosphorylation of Phogrin by PKA serves to inactivate Phogrin at the PM so that it does not hydrolyze PIPs in the PM and endosome during exocytosis and vesicle recycling. Aim 4. Use knockdown of endogenous Phogrin to test the hypothesis that endogenous Phogrin is a determinant of DCV PI(4,5)P2 content and plays a role in secretagogue-stimulated exocytosis. These studies will provide new information toward the understanding of insulin secretion and push forward our knowledge as to what points of the insulin release secretory pathway may be mis-regulated in diabetes. Further, I expect to obtain data which will guide my future studies toward clinical treatments to correct aberrant steps in insulin secretion.